Cerebral ischemia, also termed “stroke” or “brain attack,” is a leading cause of mortality and neurologic disability worldwide, but proven treatment options are severely limited. Recent clinical results indicate that the timely administration of thrombolytic agents can improve the outcome from stroke by restoring blood flow to the ischemic brain, for instance, but this approach has its limitations and additional therapeutic modalities are urgently needed (Anonymous, N Engl J Med 333:1581–1587, 1995). The timing of therapeutic intervention against stroke damage is critical because outside the most profoundly ischemic zone where all cells are destined to die (the “ischemic core”), lies a “penumbral zone” where brain cell death slowly continues to occur for minutes, hours and days after the onset of ischemia. Delayed cell death in the under-perfused penumbral region is caused by a poorly understood cascade of cytotoxic mediators that kill otherwise potentially viable cells. The time course of progressive brain damage within this penumbra limits the duration of the therapeutic window, and new therapeutic approaches will depend first on the identification of responsible cytotoxic mediators and secondly on the identification of antagonists that can be administered within the therapeutic window. Thus the goal of therapy for cerebral infarction is to prevent the loss of potentially viable brain tissue in the early hours after the onset of ischemia, and there is a need to identify both target mechanisms of cytotoxicity and suitable antagonist agents to minimize brain damage in stroke.
alpha-2HS-Glycoprotein (α2-HS), sometimes called human fetuin, is the human homolog of the bovine protein originally isolated as fetuin. Alpha-2HS-Glycoprotein is a major protein occurring in human blood and calciferous tissues (where has been known as “bone resorptive protein-2,” or BRP-2). Due to extensive sequence identity, α2-HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native α2-HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N-glycosylations and O-glycosylations, sulfation of the carbohydrate side chains, and phosphorylation, such that slightly differing mature forms may be present. α2-HS is generally considered to comprise two polypeptide chains, the A chain (282 amino acids) with five internal disulfide bridges forming it into a series of loops, and the B chain (27 amino acids) linked by a single disulfide bridge to the A chain. Human fetuin, or α2-HS, is generally considered to arise from a single mRNA transcript encoding a 367 amino acid peptide known as the “alpha-2-HS-glycoprotein precursor” (SEQ ID NO. 1). Amino acids 1–18 (SEQ ID NO. 2) comprise a signal sequence domain. Amino acids 19–300 comprise the α2-HS-glycoprotein A chain domain (SEQ ID NO. 3). Amino acids 341–367 comprise the α2-HS-glycoprotein B chain domain (SEQ ID NO. 4). By inference, amino acids 301–340 comprise a 40 amino acid connecting sequence (SEQ ID NO. 5) that is not present in the mature form, although single chain forms of α2-HS have been isolated (Jahnen-Dechent et al., Eur. J Biochem. 226:59–69, 1994).
Fetuin was first identified more than 50 years ago as a major protein component of bovine fetal serum but its biological function remains unclear, particularly as a circulating protein. Bovine fetuin occurs as a single chain, globular 341 amino acid polypeptide (amino acids 19–359 of the 359 amino acid bovine fetuin precursor) with six internal disulfide bonds and three N-linked and two O-linked oligosaccharides (SEQ ID NO. 6). Primary amino acid sequence and the position of cysteine residues are well conserved across species, e.g., human, bovine, sheep, rat and mouse (Dziegielewska et al., J. Biol. Chem. 265:4354, 1990; Rauth et al., Euro J. Biochem. 205:321, 1992; Lee et al., Proc. Natl. Acad. Sci. USA 84:4403, 1987; and Brown et al., Eur. J. Biochem. 205:321, 1992). Fetuin (α2-HS) levels in human plasma are regulated in the manner of a negative acute phase reactant (Lebreton et al., J. Clin. Invest. 64:1118, 1979). IL-1 was shown to suppress α2-HS transcript levels in cultured hepatocytes (Akhoundi et al., J. Biol. Chem. 268:15925, 1994). α2-HS appears to be expressed in bone because transcripts have been detected in both chondrocytes and osteoblasts (Yang et al., Blood 12:7, 1991), and α2-HS influences the mineral phase of bone. The α2-HS glycoprotein is the human homolog of fetuin and is secreted in high levels by adult liver into the peripheral circulation (Triffitt et al., Nature 262:226, 1976).
Human fetuin (α2-HS) has 2 N-linked oligosaccharide chains (attached to the amine nitrogen atom of asparagine), and 3 O-linked oligosaccharide chains (attached to the oxygen atom of serine or threonine). The sugar moiety directly attached to the α2-HS polypeptide is usually a N-acetylglucosamine residue. The terminal sugar residue is usually a sialic acid, in particular a N-acetylneuraminic acid (NANA) residue, which bears a net negative charge. If one removes the terminal sialic acid residue from α2-HS by neuraminidase treatment, the resulting glycoprotein is an asialofetuin. Fetuin (α2-HS) is also a carrier protein for growth factors and cytokines. The synthesis of human α2-HS-glycoprotein is down-regulated by cytokines (hIL-1β, hIL-6) (Lebreton et al., J. Clin. Invest. 64:1118–1129, 1979). Human fetuin (α2-HS) levels are decreased (25–50%) in trauma patients (van Oss et al., J. Trauma 15:451, 1975). α2-HS is structurally related to the cystatins and kininogens.